A fusion protein and a double subunit vaccine simultaneously expressing BVDV E2 protein and BRSV F protein

Abstract

The application provides a fusion protein and a double subunit vaccine simultaneously expressing BVDV E2 protein and BRSV F protein, and relates to the technical field of animal biological products.The fusion protein is subjected to enzymatic hydrolysis and in-vitro refolding to obtain a bovine viral diarrhea and bovine respiratory syncytial virus double subunit vaccine with complete structure and function, and the bovine viral diarrhea virus E2 protein is in the form of a dimer, and the bovine respiratory syncytial virus F protein is in the form of a trimer to form the vaccine.The application realizes the production of two kinds of virus subunit vaccines simultaneously in one expression process, the purification filler and the enzyme for enzymatic hydrolysis can be reused, the purification process is simple, the production cost is low, the antigen purity is high, no ADE effect is generated, and the immune side reaction is effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of veterinary biological products, and particularly relates to a fusion protein simultaneously expressing BVDV E2 protein and BRSV F protein and a double subunit vaccine. BACKGROUND

[0002] Bovine viral diarrhea (BVD) is a disease characterized by mucosal inflammation, erosion, necrosis and diarrhea in cattle caused by bovine viral diarrhea virus (BVDV). Under natural conditions, this disease can infect domestic and wild ruminants, but mainly affects 6-18 month-old calves. Sick cattle show sudden onset, body temperature rises to 40-42℃, loss of appetite, severe damage to the digestive tract mucosa, and initial symptoms of watery diarrhea, followed by blood and mucosa in the stool. The mortality rate of sick cattle can be as high as 90%.

[0003] The BVDV genome is a single-stranded positive RNA, with a full length of about 12.5Kb, containing only one ORF that encodes about 4000 amino acid polyproteins. After translation and processing, the polyproteins can form 11 mature proteins, of which C, Erns, E1, E2 are structural proteins of the virus. E2 is the envelope glycoprotein of BVDV, with the strongest immunogenicity, capable of inducing both humoral and cellular immunity in the body, and producing neutralizing antibodies, so it is the preferred gene for preparing detection antigens and vaccines.

[0004] Bovine respiratory syncytial (BRS) is an acute, febrile respiratory tract infectious disease caused by bovine respiratory syncytial virus (BRSV), with symptoms of high fever, cough, runny nose and salivation, etc. It is one of the main pathogens causing respiratory diseases in ruminants such as cattle. The disease is distributed worldwide and causes great harm to the cattle industry. According to reports, the morbidity of 15-18 month-old cattle is as high as 80-100%, and the mortality rate of some cattle farms is 5-20%, causing great economic losses to the cattle industry.

[0005] BRSV is a member of the family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus, is an enveloped virus with a negative-sense, single-stranded RNA genome of approximately 15 kb in length, which is transcribed into 10 viral RNAs, of which 9 are structural proteins, including G, F, SH, M, M2-1, M2-2, P, L and N; and 2 are non-structural proteins, including NS1 and NS2. G and F are the two major protective antigens of BRSV and are the main determinants of BRSV host tropism. Because of the high variability of G protein, weak cross-protection and the fact that G protein immunization can cause antibody-dependent enhancement (ADE) of disease, the G protein of BRSV is not the preferred protein for a subunit vaccine. F protein is an N-glycosylated type I transmembrane glycoprotein that mediates fusion of the viral envelope with the host cell through dramatic conformational changes. Before fusion is initiated, the F protein adopts a prefusion conformation, which is unstable with a low energy barrier. When the virus is in close proximity to the host, the fusion peptide inserts into the host cell membrane and the F protein is able to span the viral and host cell membranes. Subsequently, the F protein forms a trimeric, hairpin structure that links the two membranes together, facilitating fusion. The post-fusion conformation of the F protein is very stable. The prefusion conformation of the F protein trimer is "lollipop" shaped, and the post-fusion conformation of the F protein is "walking stick" shaped. The two conformations are very different in structure and have different antigenic epitopes. Ideally, to prevent viral entry, a vaccine needs to be developed with the antigenic epitopes of the prefusion conformation of the F protein. Due to the stability of the post-fusion conformation, the complete F protein obtained by a recombinant expression system can only maintain the stable post-fusion conformation. Therefore, we made a series of genetic mutations to the gene coding sequence of the F protein, removed the transmembrane region and intracellular region at the C-terminus of the F protein, and introduced a trimer structure stabilizing motif at the C-terminus. The Furin protease site and the transmembrane region were replaced with a flexible "GSGSGR" sequence, so as to stabilize the prefusion conformation. There is no specific treatment method at present, and symptomatic treatment and intensive care can alleviate symptoms, enhance the body's resistance, and promote the recovery of sick cattle. Commercial vaccines for BVDV and BRSV include inactivated vaccines and attenuated vaccines, but both types of vaccines have shortcomings. BVDV attenuated vaccine can cause abortion in cows and lead to persistent infection in cattle; the inactivated vaccine is safe, but the antibody titer level produced after vaccination is lower than that of the attenuated vaccine. BRSV inactivated vaccine can also cause ADE effect, thereby enhancing the infectivity of the virus; the attenuated vaccine has poor immunization effect in the presence of maternal antibodies and is not conducive to virus purification. Therefore, there is an urgent need to develop a new type of vaccine that has strong immunogenicity, does not produce ADE effect, is safe, has low cost, and can prevent the above two diseases at the same time. SUMMARY

[0006] In view of the above, the present application aims to provide a fusion protein and a bivalent subunit vaccine expressing BVDV E2 protein and BRSV F protein simultaneously, wherein the BVDV E2 and BRSV F protein are connected by a TEV enzyme cutting motif to form a fusion protein, and the subunit vaccine of bovine viral diarrhea and bovine respiratory syncytial virus disease with complete structure and function can be obtained through enzyme cutting and in vitro refolding by using a one-time production process.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0008] The present application provides a fusion protein expressing bovine viral diarrhea virus E2 protein and bovine respiratory syncytial virus F protein simultaneously, wherein the structure of the fusion protein comprises, from N-terminal to C-terminal, a signal peptide, bovine viral diarrhea virus E2 protein, bovine IgG Fc, a TEV enzyme cutting motif, bovine respiratory syncytial virus F protein and Fibritin trimerization sequence.

[0009] Preferably, the source of the signal peptide comprises mouse IgG kappa chain signal peptide and / or Bombyx mori immunoglobulin signal peptide.

[0010] The source of the Fc comprises bovine IgG Fc.

[0011] The present application also provides a fusion protein expressed by CHO cells, wherein the structure of the fusion protein from N-terminal to C-terminal comprises, in sequence, mouse IgG kappa chain signal peptide, bovine viral diarrhea virus E2 protein, bovine IgG Fc, a TEV enzyme cutting motif, bovine respiratory syncytial virus F protein and Fibritin trimerization sequence.

[0012] The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown in SEQ ID NO. 1, the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO. 2, the nucleotide sequence of the bovine IgG Fc is shown in SEQ ID NO. 3, the nucleotide sequence of the TEV enzyme cutting motif is shown in SEQ ID NO. 4, the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown in SEQ ID NO. 5, and the nucleotide sequence of the Fibritin trimerization sequence is shown in SEQ ID NO. 6.

[0013] Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 8.

[0014] The application further provides the fusion protein expressed by an insect cell, and the structure of the fusion protein from N-terminal to C-terminal comprises in sequence: a Bombyx mori immunoglobulin signal peptide, a bovine viral diarrhea virus E2 protein, a bovine IgG Fc, a TEV enzyme cutting motif, a bovine respiratory syncytial virus F protein and a Fibritin trimerization sequence.

[0015] The nucleotide sequence of the Bombyx mori immunoglobulin signal peptide is shown as SEQ ID NO. 9, the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown as SEQ ID NO. 10, the nucleotide sequence of the bovine IgG Fc is shown as SEQ ID NO. 11, the nucleotide sequence of the TEV enzyme cutting motif is shown as SEQ ID NO. 12, the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown as SEQ ID NO. 13, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID NO. 14.

[0016] Preferably, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 16.

[0017] Preferably, the fusion protein is secreted in the culture supernatant after the signal peptide is removed, and the amino acid sequence after the signal peptide is removed is shown as SEQ ID NO. 17.

[0018] The application provides a recombinant vector comprising the fusion protein.

[0019] The application provides a recombinant cell line for expressing the fusion protein.

[0020] The application further provides application of the fusion protein, the recombinant vector or the recombinant cell line in preparation of a bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine.

[0021] The application further provides a bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine, and the fusion protein expressed by the recombinant cell line is used as an antigen.

[0022] Beneficial effects: the application provides a fusion protein simultaneously expressing BVDV E2 protein and BRSV F protein, wherein the BRSV F protein is modified before the fusion protein, including deleting the Furin protease site and P27 sequence from 109th to 136th, and replacing the flexible "GSGSGR" sequence, and in order to enhance the trimerization of monomers, removing the transmembrane region and intracellular region from 526th to 574th at the C terminal, and replacing with a Fibritin trimerization sequence, so that the complete function of the F protein can be realized, and the fusion protein is not broken before being secreted from the cell due to the hydrolysis of the Furin protease. Finally, the BVDV E2 and BRSV F protein are connected by a TEV enzyme cutting motif to form a fusion protein, and a bovine viral diarrhea and bovine respiratory syncytial virus disease subunit vaccine with complete structure and function is obtained through a one-time production process, enzyme cutting and in-vitro refolding.

[0023] The fusion protein described in the application adds an FC tag, promotes the expression of bovine viral diarrhea virus E2 protein in the form of dimers, prolongs the half-life of the protein, promotes antigen presentation, and after one-time expression, a bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine can be produced at the same time, and finally the bovine viral diarrhea virus E2 protein is in the form of dimers, and the bovine respiratory syncytial virus F protein is in the form of trimers to form the vaccine. The commercial Protein A filler can be used to purify the fusion protein, the purification process is simple, the antigen purity of the vaccine is high, no ADE effect is produced, and the immune side reaction is effectively reduced. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 For determining the BVDV antibody titer before and after immunization by using the IDXEE kit;

[0025] Figure 2 For detecting the BRSV antibody titer by using the F protein of BRSV to coat the ELISA plate. DETAILED DESCRIPTION

[0026] The application provides a fusion protein simultaneously expressing bovine viral diarrhea virus E2 protein and bovine respiratory syncytial virus F protein, wherein the structure of the fusion protein comprises, from the N terminal to the C terminal, a signal peptide, bovine viral diarrhea virus E2 protein, Fc, a TEV enzyme cutting motif, bovine respiratory syncytial virus F protein and a Fibritin trimerization sequence.

[0027] The source of the signal peptide preferably includes a mouse IgG kappa chain signal peptide and / or a Bombyx mori immunoglobulin signal peptide; and the source of the Fc preferably includes a bovine IgG Fc. In the present application, the BRSV F protein, before being fused, preferably further includes modification, and more preferably includes deletion of a Furin protease site at positions 109 to 136 and a P27 sequence, and replacement of a flexible "GSGSGR" sequence, and in order to enhance trimerization of monomers, removal of a transmembrane region and intracellular region at positions 526 to 574 at the C terminal, and replacement with a Fibritin trimerization sequence, so that the complete function of the F protein can be realized, and the fusion protein is not broken before being secreted from the cell due to the hydrolysis of the Furin protease, the complexity of protein folding is reduced, and the structural stability is increased.

[0028] The fusion protein of the present application needs to be codon-optimized according to the codon bias of the expression cell when expressed by different cell lines, so that the nucleotide sequences of the fusion protein are slightly different based on different cell expression systems, although the structure of the fusion protein is the same.

[0029] The present application also provides a fusion protein expressed by CHO cells, and the structure of the fusion protein from the N terminal to the C terminal includes, in sequence, a mouse IgG kappa chain signal peptide, a bovine viral diarrhea virus E2 protein, a bovine IgG Fc, a TEV enzyme cleavage motif, a bovine respiratory syncytial virus F protein, and a Fibritin trimerization sequence.

[0030] The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown as SEQ ID NO. 1, the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown as SEQ ID NO. 2, the nucleotide sequence of the bovine IgG Fc is shown as SEQ ID NO. 3, the nucleotide sequence of the TEV enzyme cleavage motif is shown as SEQ ID NO. 4, the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown as SEQ ID NO. 5, and the nucleotide sequence of the Fibritin trimerization sequence is shown as SEQ ID NO. 6.

[0031] The fusion protein expressed by the CHO cell in the present application is referred to as CHO-E2-F sequence, which is synthesized by codon optimization referring to the E2 gene sequence of bovine viral diarrhea virus 3877 strain (MW013505) and the F gene sequence of bovine respiratory syncytial virus ATCC51908 strain (NC038272) in the examples, but it cannot be identified as the whole protection scope of the present application. The CHO-E2-F sequence comprises, in order, mouse IgG kappa chain signal peptide with 63 bases, the nucleotide sequence of which is shown in SEQ ID NO. 1; BVDV E2 protein sequence with 1017 bases, the nucleotide sequence of which is shown in SEQ ID NO. 2; bovine IgG Fc sequence with 696 nucleotides, the nucleotide sequence of which is shown in SEQ ID NO. 3; TEV enzyme cutting motif with 21 bases, the nucleotide sequence of which is shown in SEQ ID NO. 4; BRSV F protein sequence with 1434 bases, the nucleotide sequence of which is shown in SEQ ID NO. 5; Fibritin trimerization sequence with 75 bases, the nucleotide sequence of which is shown in SEQ ID NO. 6. After synthesizing the synthetic CHO-E2-F sequence, the present application preferably further comprises setting HindIII and EcoRI enzyme cutting sites at both ends of the CHO-E2-F sequence, with a full length of 3327 bases, the nucleotide sequence of which is shown in SEQ ID NO. 7, and the encoded amino acid sequence is shown in SEQ ID NO. 8.

[0032] The present application also provides a fusion protein expressed by an insect cell, which comprises, in order from N-terminal to C-terminal, silkworm immunoglobulin signal peptide, bovine viral diarrhea virus E2 protein, bovine IgG Fc, TEV enzyme cutting motif, bovine respiratory syncytial virus F protein and Fibritin trimerization sequence.

[0033] The nucleotide sequence of the silkworm immunoglobulin signal peptide is shown in SEQ ID NO. 9, the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO. 10, the nucleotide sequence of the bovine IgG Fc is shown in SEQ ID NO. 11, the nucleotide sequence of the TEV enzyme cutting motif is shown in SEQ ID NO. 12, the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown in SEQ ID NO. 13, and the nucleotide sequence of the Fibritin trimerization sequence is shown in SEQ ID NO. 14.

[0034] The present application does not have special limitation on the kind of the insect cell, and the insect cell SF9 is taken as an example in the embodiment, but it cannot be only identified as the whole protection scope of the present application. In the embodiment of the present application, the sequence of the fusion protein, referred to as SF9-E2-F sequence, is synthesized by referring to the sequence of E2 gene of bovine viral diarrhea virus 3877 strain (MW013505) and the sequence of F gene of bovine respiratory syncytial virus ATCC51908 strain (NC038272) and codon optimization. The SF9-E2-F sequence of the present application preferably comprises, in sequence, the sequence of the signal peptide of Bombyx mori immunoglobulin, having a size of 60 bases, the nucleotide sequence of which is shown as SEQ ID NO. 9; the sequence of BVDV E2 protein, having a size of 1017 bases, the nucleotide sequence of which is shown as SEQ ID NO. 10; the sequence of bovine IgG FC, having a size of 696 nucleotides, the nucleotide sequence of which is shown as SEQ ID NO. 11; the TEV enzyme cutting motif, having a size of 21 bases, the nucleotide sequence of which is shown as SEQ ID NO. 12; the sequence of BRSV F protein, having a size of 1434 bases, the nucleotide sequence of which is shown as SEQ ID NO. 13; the Fibritin trimerization sequence, having a size of 75 bases, the nucleotide sequence of which is shown as SEQ ID NO. 14; and the synthesized SF9-E2-F sequence contains the BamHI and HindIII enzyme cutting sites at both ends, and has a full length of 3324 bases, the nucleotide sequence of which is shown as SEQ ID NO. 15, and the encoded amino acid sequence is shown as SEQ ID NO. 16.

[0035] The CHO-E2-F sequence or the SF9-E2-F sequence of the present application is secreted in the culture supernatant after the signal peptide is removed, and the amino acid sequence after the signal peptide is removed is shown as SEQ ID NO. 17.

[0036]

[0037] The present application provides a recombinant vector containing the fusion protein.

[0038] In the present application, the recombinant vector constructed in the middle is also different for different expression cells. When the fusion protein is CHO-E2-F sequence, the CHO-E2-F sequence and pEE12.4 vector are preferably cleaved by HindIII and EcoRI, and the cleavage fragments are recovered and ligated to transform clones, and sequencing is correct, so that the CHO-E2-F-pEE12.4 recombinant plasmid is constructed. When the fusion protein is SF9-E2-F sequence, the SF9-E2-F sequence and pFastBac1 vector sequence are preferably cleaved by BamHI and HindIII, respectively, and the cleavage fragments are recovered and ligated to transform clones, and sequencing is verified, so that the SF9-E2-F-pFastBac1 transfer vector is constructed.

[0039] The present application provides a recombinant cell line expressing the fusion protein.

[0040] The present application does not have special limitations on the construction method of the recombinant cell line, and the conventional construction method in the art can be used. After the CHO-E2-F-pEE12.4 recombinant plasmid is constructed, CHO-K1 cell transfection and high expression cell strain screening are used, so that a stable transfection cell strain (high expression cell strain) is screened, and the high expression cell strain is serum-free suspension domesticated to obtain the recombinant cell line. The present application preferably performs cell shake flask fermentation on the recombinant cell line, harvests the cell supernatant after fermentation, and purifies the protein by using commercial protein A filler. After the purified protein is obtained, the present application preferably further includes the step of adding 1 UTEV protease per 8 μg of protein for enzymatic cleavage, loading the cleaved protein into a dialysis bag for VLPs self-assembly, taking out the liquid in the dialysis bag, loading on a nickel column, removing the TEV enzyme containing His tag, and the flow-through is the antigen solution containing BVDV E2 protein dimer and BRSV F protein trimer, which can be applied to prepare a BVDV and BRSV double subunit vaccine.

[0041] After obtaining the SF9-E2-F-pFastBac1 transfer vector, the application preferably further comprises transforming the competent cell DH10Bac, culturing at 37 DEG C, screening by using blue-white spot screening method and performing PCR identification to obtain the SF9-E2-F-Bacmid recombinant bacmid; and transfecting the SF9-E2-F-Bacmid recombinant bacmid into the SF9 cells in logarithmic growth phase, harvesting the P1 generation of recombinant baculovirus SF9-E2-F-rBV after culturing, and continuously passing to P3 generation, centrifuging the P3 generation virus, and taking the supernatant as the virus liquid; infecting the HighFive cells with the P3 generation virus, culturing, and collecting the supernatant, wherein the supernatant contains the target protein. The purification, enzyme cutting and antigen liquid preparation method of the target protein are the same, and will not be repeated here.

[0042] The application also provides application of the fusion protein, the recombinant vector or the recombinant cell line in preparation of a bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine.

[0043] The application is preferably the same as described above, and will not be repeated here.

[0044] The application also provides a bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine, wherein the fusion protein expressed by the recombinant cell line is used as an antigen.

[0045] After obtaining the antigen liquid, the application preferably further comprises diluting the antigen liquid with PBS buffer to a final concentration of 60 μg / mL, mixing and emulsifying the antigen liquid with the ISA 201 VG adjuvant according to a volume ratio of 46:54, so as to obtain the double subunit vaccine.

[0046] The fusion protein and the double subunit vaccine for simultaneously expressing BVDV E2 protein and BRSV F protein are described in detail in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0047] In the examples of the application, the reagents and materials used are all conventional commercially available products in the art, for example, the BVDV virus (W strain) is purchased from the China Veterinary Drug Inspection Institute, and the BRSV is purchased from the American Type Culture Collection (ATCC), and the strain is ATCC51908.

[0048] Example 1

[0049] Sequence synthesis

[0050] Based on the E2 gene sequence of bovine viral diarrhea virus strain 3877 (MW013505) and the F gene sequence of bovine respiratory syncytial virus strain ATCC51908 (NC038272), the CHO-E2-F sequence was synthesized after codon optimization. The synthesized CHO-E2-F sequence contains HindIII and EcoRI restriction sites at both ends and has a full length of 3327 bases, as shown in SEQ ID NO.7. The amino acid sequence encoded by the CHO-E2-F sequence is shown in SEQ ID NO.8.

[0051] Example 2

[0052] Construction of CHO-E2-F-pEE12.4 recombinant plasmid

[0053] The CHO-E2-F sequence and pEE12.4 vector (purchased from Jiman Biotechnology) synthesized in Example 1 were digested with HindIII and EcoRI. The digested CHO-E2-F and pEE12.4 sequence fragments were recovered, ligated, transformed, and cloned. After sequencing verification, the CHO-E2-F-pEE12.4 recombinant plasmid was successfully constructed.

[0054] Example 3

[0055] CHO-K1 cell transfection and screening of high-expressing cell lines

[0056] 1. Prepare cells:

[0057] Take one 10cm cell culture dish of CHO-K1 cells that have been passaged for 24 hours and have a confluence of 80% to 90%. Discard the culture medium, wash the cells once with 10mL PBS, discard the PBS, add 15mL of serum-free and antibiotic-free DMEM / F12 medium, and incubate in a 37℃ 5% CO2 cell culture incubator.

[0058] 2. Transfection plasmid

[0059] according to According to the 2000 instructions, 24 μg of the CHO-E2-F-pEE12.4 recombinant plasmid and 60 μL of serum-free and antibiotic-free DMEM / F12 medium were diluted separately. 2000, mix well and let stand at room temperature for 5 minutes. Mix the two tubes of liquid and let stand at room temperature for 20 minutes. Take the prepared CHO-K1 cells, add the above-mentioned mixed reagent dropwise, and then place them in a 37℃ 5% CO2 cell culture incubator. After culturing for 4-6 hours, discard the culture medium, add 10 mL of DMEM / F12 medium (10% serum, 1% penicillin and streptomycin, the same below, glutamine-free), and place in a 37℃ 5% CO2 cell culture incubator.

[0060] 3. Pressure selection

[0061] 24h after transfection, take one dish of transfected CHO-K1 cells and one dish of untransfected CHO-K1 cells (negative control), discard the supernatant medium, add 10 mL DMEM / F12 medium (10% serum + 25 μM MSX, 1% double antibody, without glutamine), pressure selection for 7 days, observe the cells in the middle, when there are many dead cells, change the medium to the same medium.

[0062] 4. Screening of stable cell strain

[0063] When the negative control cells are basically dead after 25 uM MSX pressure selection for about 7 days, start cell strain screening. Take the transfected CHO-K1 cells, discard the medium, wash once with PBS, add 500 μL 0.25% trypsin, digest for 2-5 min at room temperature, digest until the cells are single cells, add 10 mL DMEM / F12 medium (10% serum + 25 uM MSX, 1% double antibody, without glutamine) to stop the digestion reaction, blow the cells with a pipette, and count the cells. Dilute the cells to 10 4 cells / mL with DMEM / F12 medium (10% serum + 25 μM MSX, 1% double antibody, without glutamine), transfer to a 96-well plate, 200 μL per well, and place in a 37°C 5% CO2 cell incubator for culture. When the 96-well plate is full, take the supernatant, ELISA detection, and continue to culture and freeze the high-expression positive cell strain.

[0064] Example 4

[0065] Serum-free suspension domestication of high-expression cell strain

[0066] Recover the high-expression cell strain in DMEM / F12 medium (10% serum, 1% double antibody, without glutamine), and continue to use this medium for 2-3 passages until the cell growth is stable. When the cell density reaches 2 x 10 6 cells / mL, passage the cells at a density of 0.3-0.5 x 10 5 cells / mL, and the medium is a mixture of 10% serum DMEM / F12 medium and base medium (purchased from Jianshun Biology) at a ratio of 75:25. Culture the cells in a constant temperature shaker at 37°C, 5% CO2, and a rotation speed of 110 rpm. When the cell density reaches 2 x 10 6 cells / mL and the cell viability is >90%, passage the cells at a density of 0.3-0.5 x 10 cells / mL, and the medium is a mixture of 10% serum DMEM / F12 medium and 6cells / mL. If the cell growth is slow, centrifuge the supernatant, leave 20% of the original medium, and add 80% of the 10% serum DMEM / F12 medium to the supernatant The basic medium ratio is 75:25 of the mixed medium, and the culture is continued; the foregoing steps are repeated, and the proportion of the basic medium is gradually increased to 100% of the basic medium.

[0067] Example 5

[0068] Cell flask fermentation

[0069] The flask cells are taken out from the constant temperature shaker, and the cells are diluted to 0.3-0.5 x 10 5 cells / mL. If the cell growth is slow, centrifuge the supernatant, leave 20% of the original medium, and add 80% of the 10% serum DMEM / F12 medium to the supernatant The basic medium ratio is 75:25 of the mixed medium, and the culture is continued; the foregoing steps are repeated, and the proportion of the basic medium is gradually increased to 100% of the basic medium.

[0070] Example 6

[0071] Insect cell expression sequence synthesis

[0072] Referring to the Bovine viral diarrhea virus 3877 strain E2 gene sequence (MW013505) and the Bovine respiratory syncytial virus ATCC51908 strain F gene sequence (NC038272), the SF9-E2-F sequence is synthesized by codon optimization. The synthesized SF9-E2-F sequence contains BamHI and HindIII enzyme cutting sites at both ends, and the full length is 3324 bases, as shown in SEQ ID NO. 15, and the encoded amino acid sequence is shown in SEQ ID NO. 16.

[0073] Example 7

[0074] Baculovirus transfer vector construction

[0075] The SF9-E2-F sequence synthesized in Example 6 and the pFastBac1 vector sequence are cut by BamHI and HindIII, the cut SF9-E2-F and pFastBac1 sequence fragments are recovered, and the ligation transformation cloning is performed. After sequencing verification, no error is found, and the SF9-E2-F-pFastBac1 transfer vector is constructed.

[0076] Example 8

[0077] Bacmid preparation​

[0078] The SF9-E2-F-pFastBac1 transfer vector prepared in Example 7 was transformed into competent DH10Bac cells. After culturing at 37°C, the cells were screened using the blue-white screening method and identified by PCR to obtain SF9-E2-F-Bacmid recombinant rod granules. The rod granule transformation and screening methods were based on those described in Invitrogen's Bac-to-Bac... TM Follow the user guide for the baculovirus expression system.

[0079] Example 9

[0080] Recombinant baculovirus harvest

[0081] The SF9-E2-F-Bacmid recombinant rod-like particles obtained in Example 8 were transfected using a transfection reagent. SF9 cells in logarithmic growth phase were transfected, and after 72 hours of culture, P1 generation recombinant baculovirus SF9-E2-F-rBV was harvested. The harvested P1 generation recombinant baculovirus was passaged in SF9 cells to the P3 generation. The P3 generation virus was centrifuged, and the supernatant was used as the viral fluid. The viral titer of the P3 generation was determined by plaque assay. HighFive cells were infected with the P3 generation virus at an inoculum size of 1 MOI, and after 96 hours of culture, the culture supernatant contained the target protein.

[0082] Example 10

[0083] Protein purification

[0084] Collect the cell supernatant from Example 5 or 9, centrifuge at 8000g for 30 min at 4°C, collect the supernatant, and filter through a 0.8 μm filter membrane. Equilibrate the Protein A column with 5–10 column volumes of PBS. Repeat the column loading with the treated cell supernatant 3 times. Wash the column with 10-fold PBST, wash with 2-fold PBS, and elute the protein with 2 column volumes of 0.1M glycine (pH 3.0). Collect the eluent and neutralize to pH 7.5 with 1M Tris (pH 9.0). This is the purified protein.

[0085] Example 11

[0086] Protein cleavage and refolding

[0087] The purified protein concentration was determined according to the BCA protein quantitative detection kit (purchased from Shanghai Biotech) instructions, the total protein mass was calculated, 1 U TEV protease (His-tag, purchased from Biyun Tian Bio) was added per 8 μg of enzyme digestion protein, 10x enzyme digestion buffer (500 mM NaH2PO4, 150 mM NaCl, 10 mM EDTA, 10 mM DTT, 1% Tween-20, pH 8.0) was added by volume, and enzyme digestion was performed at 4°C for 12-16 h. After enzyme digestion was completed, the enzyme digestion protein was loaded into a dialysis bag (3500D), the dialysis solution was 50 mM NaH2PO4, 500 mM NaCl, pH 8.0, and dialysis was performed for 12-16 h, with 2-3 changes of dialysis solution during the period. After the first dialysis was completed, the protein was allowed to stand at 4°C for 8-12 h to complete the protein refolding. The dialysis solution was replaced with PBS buffer, and dialysis was performed for 12-16 h, with 2-3 changes of dialysis solution during the period. After the second dialysis was completed, the liquid in the dialysis bag was removed, loaded onto a nickel column, and the TEV enzyme containing His-tag was removed, and the flow-through was the antigen solution containing BVDV E2 protein dimer and BRSV F protein trimer. The nickel column was eluted with PBS buffer containing 200 mM imidazole, and the eluate was collected, which was the recovered TEV protease, which could be directly used for enzyme digestion in the next production process after enzyme activity determination.

[0088] Example 12

[0089] Vaccine preparation

[0090] The antigen solution of Example 11 was diluted with PBS buffer to a final concentration of 60 μg / mL, mixed and emulsified with ISA 201 VG adjuvant at a volume ratio of 46:54, and the bovine viral diarrhea and bovine respiratory syncytial virus disease double-subunit vaccine was obtained.

[0091] Example 13

[0092] F protein coated ELISA plate to detect BRSV antibody

[0093] PBS buffer equilibrated Protein A column, the antigen solution containing BVDV E2 protein dimer and BRSV F protein trimer as described in Example 11 was loaded 3 times, and the flow-through was the antigen solution containing only BRSV F protein. The concentration was quantified by BCA method, and the antigen solution was diluted to 0.2 μg / mL with PBS buffer, 100 μL / well, and coated at 4°C for 16 h. After coating, the liquid in the wells was discarded, 300 μL of PBST washing solution was added to each well, and the wells were rinsed once. 200 μL of freshly prepared blocking solution (5% skim milk, PBS) was added to each well, and the wells were blocked at 37°C for 2 h. After blocking, the liquid in the wells was discarded, 300 μL of PBST washing solution was added to each well, and the wells were rinsed once and dried on a water-absorbing filter paper. The BRSV serum to be tested was diluted 100-fold with PBS buffer, added to the antigen-coated plate, and incubated at 37°C for 1 h. The liquid in the wells was discarded, 300 μL of washing solution was added to each well, and the wells were rinsed 3 times. The HRP-labeled rabbit anti-bovine IgG Fab secondary antibody diluted 10,000-fold with PBS containing 5% skim milk was added to each well at 100 μl, and the wells were incubated at 37°C for 1 h. The liquid in the wells was discarded, 300 μL of PBST washing solution was added to each well, and the wells were rinsed 3 times. 100 μL of TMB color developing solution was added to each well, and the wells were developed at room temperature for 10 min in the dark. 50 μL of stop solution was added to each well, and the absorbance at 450 nm was read on a microplate reader. The antibody positive standard was judged as follows: P / N≥2.1, OD450≥0.4.

[0094] Example 14

[0095] Immune experiment

[0096] Seven 4-5 month-old calves (BVDV and BRSV antibody negative) were randomly divided into two groups, 5 calves in the vaccine immunization group and 2 calves in the blank control group. The calves in the vaccine immunization group were immunized with bovine viral diarrhea and bovine respiratory syncytial virus disease double subunit vaccine, and the calves in the blank control group were immunized with PBS. Each calf was injected intramuscularly with 1 ml each time, and the calves were boosted once after three weeks of priming. The serum was collected before immunization, 21 days after the first immunization, and 21 days after the second immunization. The BVDV antibody titers before and after immunization were determined using the IDEXX BVDV total antibody detection kit (item number 99-44000). The results are shown in Table 1. Figure 1 Table 1: BVDV antibody titers before and after immunization. Numbers 1 to 5 are the vaccine immunization group, and numbers 6 and 7 are the blank group. The results show that the BVDV antibody S / P values of both the pre-immunization and the blank group were less than 0.5, and both were negative. The BVDV antibody S / P values of the vaccine immunization group were greater than 0.5 21 days after the first immunization, and the BVDV antibody S / P values were around 1.5 21 days after the second immunization. The BRSV antibody titers were detected by the indirect ELISA method of Example 16, and the results are shown in Table 2. Figure 2, numbers 1 to 5 are vaccine immunization groups, and numbers 6 and 7 are blank groups. The results show that the P / N values of BRSV antibodies of the pre-immunization and blank groups are about 1, less than 2.1, and are negative. The P / N values of BVDV antibodies of the vaccine immunization groups reach above 4 after 21 days of the first immunization, and reach up to 11 after 21 days of the second immunization. The detection results of BVDV and BRSV antibodies show that the bovine viral diarrhea and bovine respiratory syncytial virus double-subunit vaccine has good immunogenicity.

[0097] Example 15

[0098] Neutralizing antibody experiment

[0099] One day before the experiment, the MDBK cells were digested with trypsin, resuspended with DMEM containing 10% FBS, and plated into 96-well cell culture plates at a cell seeding density of 2 x 10 5 cells / mL, 0.1 mL per well, and incubated at 37°C, 5% CO2. The serum sample 21 days after the second immunization was diluted with DMEM containing 2% FBS at dilutions of 1 1 , 1 2 , 1 3 , 1 4 , 1 5 , 1 6 , 1 7 , 1 8 , 1 50 . 100 μL of the serum dilution was mixed with an equal volume of BVDV virus (200 TCID 50 ) to obtain a final virus titer of 100 TCID 50 / 0.1ml, placed in 37℃, 5% CO2 incubator for 90min; take 96 well culture plate MDBK cells cultured for 24h, discard the growth solution, transfer the neutralization 90min virus and serum mixture to the corresponding hole of 96 well cell culture plate, 0.1mL / hole, different dilution serum / virus mixture is moved to the cell plate, the pipette gun head is replaced in time, 4 repeats are made for each dilution, and normal negative control cells without neutralization virus are set; placed in 37℃, 5% CO2 conditions for culture. Different dilution serum-BVDV virus mixture is inoculated with MDBK cells in the cell culture box for 72h, and the cell pathological changes are observed. If the cells have pathological changes, it means that BVDV has not been neutralized by the neutralizing antibodies produced in the serum; if the cell growth state is good, the same as the control group, it means that BVDV is neutralized by the neutralizing antibodies produced in the serum, and the cells will not appear pathological changes. The maximum dilution without CPE in MDBK cells is the neutralizing antibody titer of this serum sample. The operation of BRSV neutralizing antibody experiment is the same as above. The results of BVDV and BRSV neutralizing antibody experiment are shown in Table 1, the BVDV neutralizing antibody titer is not less than 1:128, the BRSV neutralizing antibody titer is not less than 1:64, and the neutralizing antibodies are produced in the serum 21 days after the two immunizations, which shows that the bovine viral diarrhea and bovine respiratory syncytial virus subunit vaccine can provide protection for the immunized cattle.

[0100] Table 1 BVDV and BRSV neutralizing antibody experiment results table

[0101]

[0102] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should be considered as the protection scope of the present application.

Claims

1. A fusion protein simultaneously expressing bovine viral diarrhea virus E2 protein and bovine respiratory syncytial virus F protein, characterized in that, The structure of the fusion protein, from the N-terminus to the C-terminus, includes: a signal peptide, bovine viral diarrhea virus E2 protein, Fc, TEV restriction enzyme motif, bovine respiratory syncytial virus F protein, and Fibritin trimer sequence. The signal peptide is derived from mouse IgG kappa chain signal peptide and / or silkworm immunoglobulin signal peptide; the Fc is derived from bovine IgG Fc. The amino acid sequence of the fusion protein after the signal peptide is removed is shown in SEQ ID NO.

17.

2. A fusion protein of claim 1 expressed using CHO cells, characterized in that, The structure of the fusion protein from the N-terminus to the C-terminus includes, in sequence: mouse IgG kappa chain signal peptide, bovine viral diarrhea virus E2 protein, bovine IgG Fc, TEV restriction enzyme motif, bovine respiratory syncytial virus F protein, and Fibritin trimer sequence. The nucleotide sequence of the mouse IgG kappa chain signal peptide is shown in SEQ ID NO.1; the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the bovine IgG Fc is shown in SEQ ID NO.3; the nucleotide sequence of the TEV restriction enzyme motif is shown in SEQ ID NO.4; the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown in SEQ ID NO.5; the nucleotide sequence of the Fibritin trimerization sequence is shown in SEQ ID NO.6; and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

8.

3. A fusion protein of claim 1 expressed using insect cells, characterized in that, The structure of the fusion protein from the N-terminus to the C-terminus includes, in sequence: silkworm immunoglobulin signal peptide, bovine viral diarrhea virus E2 protein, bovine IgG Fc, TEV restriction enzyme motif, bovine respiratory syncytial virus F protein, and Fibritin trimer sequence. The nucleotide sequence of the silkworm immunoglobulin signal peptide is shown in SEQ ID NO. 9; the nucleotide sequence of the bovine viral diarrhea virus E2 protein is shown in SEQ ID NO. 10; the nucleotide sequence of the bovine IgG Fc is shown in SEQ ID NO. 11; the nucleotide sequence of the TEV restriction enzyme motif is shown in SEQ ID NO. 12; the nucleotide sequence of the bovine respiratory syncytial virus F protein is shown in SEQ ID NO. 13; the nucleotide sequence of the Fibritin trimer sequence is shown in SEQ ID NO. 14; and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

16.

4. The fusion protein according to claim 2 or 3, characterized in that, The fusion protein was secreted into the culture supernatant after the signal peptide was removed, and the amino acid sequence after the signal peptide was removed is shown in SEQ ID NO.

17.

5. A recombinant vector comprising the fusion protein of claim 1, the fusion protein of claim 2 or 3, or the fusion protein of claim 4.

6. A recombinant cell line expressing the fusion protein of claim 1, the fusion protein of claim 2 or 3, or the fusion protein of claim 4.

7. The use of the fusion protein of claim 1, the fusion protein of claim 2 or 3, the fusion protein of claim 4, the recombinant vector of claim 5, or the recombinant cell line of claim 6 in the preparation of a bivalent subunit vaccine for bovine viral diarrhea and bovine respiratory syncytial virus disease.

8. A bivalent subunit vaccine for bovine viral diarrhea and bovine respiratory syncytial virus disease, characterized in that, The fusion protein expressed by the recombinant cell line of claim 6 is used as the antigen.

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